The major goal of this project is to give new directives for studies in tobacco carcinogenesis by identifying potential carcinogens in smokeless tobacco, mainstream and sidestream cigarette smoke, and environmental tobacco smoke (ETS). As in the past, newly identified suspected tobacco carcinogens (e.g. polynuclear aromatic hydrocarbons, aromatic amines, tobacco-specific N-nitrosamines) need to be quantitatively assessed, their formation has to be studied and they must be assayed in vitro and/or in vivo for tumorigenicity. We plan to investigate N-nitrosamino acids in chewing tobacco and to examine their carcinogenic activity. We will also study the fate of N-nitrosamino acids during cigarette smoking. The significant carcinogenic activities of some nitroalkanes, nitroalkenes, and protein-derived heterocyclic amines make it highly desirable to assay these pyrolysis products in tobacco smoke. We will develop new analytical methods on the basis of supercritical fluid extraction (SFE) and GC-MS. 3-Vinylpyridine, a pyrolysis product of the Nicotiana alkaloids, will be assayed as a marker that is well suited to determine the contribution of volatile tobacco smoke constituents to indoor air pollution. This far, only nicotine has been used as marker for ETS, but a reliable indicator for volatile smoke carcinogens in ETS is urgently needed. 3-Vinylpyridine appears to be suitable for this purpose, since unlike nicotine it remains static in the polluted air over a period of several hours. We have identified 4-(methylnitrosamino)-4-(3-pyridyl)butyric acid (iso- NNAC) in smokeless tobacco, but did not find it in tobacco smoke. Since iso-NNAC can be a nitrosation product of nicotine and/or its metabolites, and since it is not carcinogenic and is excreted in the urine without undergoing metabolism, it may serve as a marker for the endogenous formation of tobacco-specific N-nitrosamines (TSNA) in the urine of smokers. It is likely that Nicotiana alkaloids inhaled by the smoker are potential precursors to endogenously formed carcinogenic TSNA. Therefore, an in-depth analytical study is proposed to determine iso-NNAC in the urine of smokers. Epidemiological studies have demonstrated an increased risk for cigarette smokers for cancer of the uterine cervix. Nicotine and cotinine as well as nitrite have been identified in cervical mucus; it is likely therefore, that carcinogenic TSNA can be formed in the cervical vault of smokers. Preliminary studies utilizing highly sensitive methods, based on SFE and GC-TEA, have indicated the presence of TSNA in some samples of cervical mucus. We plan to explore this question by analyzing the cervical mucus of cigarette smokers and nonsmokers. The results of this project will answer important questions in tobacco carcinogenesis and will give impetus to new biochemical studies in chemical and environmental carcinogenesis.